The study of protein-protein interactions is an area of considerable interest. However, protein interactions are typically perturbed by traditional measurement and detection schemes. Existing methods for examining protein-protein interactions include FRET, NMR, EPR, SPR, ESI-MS, size exclusion chromatography, and native PAGE. Each of these methods requires that either one of the interacting partners is modified in some way, such as through the installation of a fluorescent label or immobilization on a surface, or that the entire complex is sieved through a matrix. These steps disrupt the transient interactions under observation, with the risk that some of the agglomerated species may be destroyed in the process of separation and labelling.
The separation and detection of components within fluid flows, such as microfluidic flows, presents a number of challenges. Given the recent increased interest in fluidic techniques for the reaction, separation and detection of components, there is interest in developing methods and devices that allow components to be separated and analysed in a continuous flow system.
The present inventors have recently described improved methods for distributing a component, including a component in a multicomponent mixture, across laminar flows in a fluidic device (see PCT/GB2013/052757). The distribution of components across the laminar flows is measured at multiple flow times by fluorescent spectroscopy. From these measurements it is possible to identify components of different size within the flows. The worked examples show the use of the methods described for the identification of Aβ(1-42) aggregation events, including the formation of oligomers and fibril clusters from the original monomeric species.
However, this work necessarily requires the use of components that are fluorescently active, or are provided with a fluorescent label. In the latter case, the behaviour of the component with the label may be affected by that label. The inventors' earlier work does not describe the purification of a component from the combined laminar flows, nor does it suggest how this might be achieved. Thus, although monomer and oligomer protein species are identified, they are not removed from the flow.
In-flow labelling and separation techniques are known in the art and have been well described by the Ramsey group (e.g. Liu et al. Anal. Chem. 2000, 72, 4608; Jacobson et al. Anal. Chem. 1994, 66, 4127; Jacobson et al. Anal. Chem. 1994, 66, 3472). For example, the group have described the electrophoretic separation of proteins on a flow device with covalent and noncovalent labelling (Liu et al.). Here, the group acknowledge the problem of labelling proteins prior to separation, particularly in electrophoretic separation experiments. Within a flow device, the group suggest downstream labelling of components after separation, rather than upstream labelling prior to separation. Electrophoretic techniques are used to draw components through the device. Here, the electrophoretic techniques separate components temporally based on their migration speed through a capillary. In this way, components having different charge-to-size ratios are distributed along the fluid flow. By way of example, the group show the separation of α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A. The efficiency of the labelling techniques is not discussed and it is nowhere suggested that the components are quantitatively labelled.
The present inventors have now established an alternative fluidic method for separating components, for example proteins in the native state, and then subsequently analysing separated components under conditions that are optimised for detection.